Benzothiazole and Chromone Derivatives as Potential ATR Kinase Inhibitors and Anticancer Agents

Despite extensive studies and the great variety of existing anticancer agents, cancer treatment remains an aggravating and challenging problem. Therefore, the development of novel anticancer drugs with a better therapeutic profile and fewer side effects to combat this persistent disease is still necessary. In this study, we report a novel series of benzothiazole and chromone derivatives that were synthesized and evaluated for their anticancer activity as an inhibitor of ATR kinase, a master regulator of the DDR pathway. The cell viability of a set of 25 compounds was performed using MTT assay in HCT116 and HeLa cell lines, involving 72 h incubation of the compounds at a final concentration of 10 µM. Cells incubated with compounds 2c, 7h and 7l were found to show viability ≤50%, and were taken forward for dose–response studies. Among the tested compounds, three of them (2c, 7h and 7l) showed higher potency, with compound 7l exhibiting the best IC50 values in both the cell lines. Compounds 2c and 7l were found to be equally cytotoxic towards both the cell lines, namely, HCT116 and HeLa, while compound 7h showed better cytotoxicity towards HeLa cell line. For these three compounds, an immunoblot assay was carried out in order to analyze the inhibition of phosphorylation of Chk1 at Ser 317 in HeLa and HCT116 cells. Compound 7h showed inhibition of pChk1 at Ser 317 in HeLa cells at a concentration of 3.995 µM. Further analysis for Chk1 and pChk1 expression was carried out in Hela cells by treatment against all the three compounds at a range of concentrations of 2, 5 and 10 µM, wherein compound 7h showed Chk1 inhibition at 2 and 5 µM, while pChk1 expression was observed for compound 7l at a concentration of 5 µM. To support the results, the binding interactions of the compounds with the ATR kinase domain was studied through molecular docking, wherein compounds 2c, 7h and 7l showed binding interactions similar to those of Torin2, a known mTOR/ATR inhibitor. Further studies on this set of molecules is in progress for their specificity towards the ATR pathway.


Introduction
Cancer remains one of the main leading causes of mortality all over the world. Despite extensive studies and the great variety of existing anticancer agents, cancer treatment remains an aggravating and challenging problem. Therefore, the development of novel

Introduction
Cancer remains one of the main leading causes of mortality all over the world. Despite extensive studies and the great variety of existing anticancer agents, cancer treatment remains an aggravating and challenging problem. Therefore, the development of novel anticancer drugs with a better therapeutic profile and fewer side effects to combat this persistent disease is still necessary.
Cells are prone to a multitude of stress factors including endogenous and exogenous factors, such as reactive oxygen species, radiation, etc., which result in genomic instability through either a single strand or a double strand break in their DNA [1]. In order to cope with the stress, and to maintain genomic integrity as well as cell viability, cells develop DNA damage response (DDR) signaling pathways which regulate and repair the damage caused [2,3]. Targeting such crucial signaling pathways in cancer cells can prevent these cells from repairing their damaged single and/or double stranded DNA, hence promoting an efficient therapeutic strategy for cancer. Rad-3 related (ATR) and ataxia telangiectasia mutated (ATM) kinases from the phosphatidylinositol-3-kinase-related kinases (PIKK) family are two major signaling proteins which phosphorylate Ser/Thr residues of their downstream substrates Chk1 and CHk2, respectively [2][3][4]. The significant role of benzothiazoles as potential kinase inhibitors generates interest in exploring the biological activity of these compounds further in the area of kinases and thus, as potential candidates for cancer therapy [5].
Benzothiazole is a heterocyclic organic compound with a wide range of biological activities such as antimicrobial [6,7], anti-inflammatory [8,9], antioxidant [10,11], analgesic [8,12], anticancer [13][14][15][16], antiviral [17][18][19][20], anti-HIV [21,22], antidiabetic [23], anticonvulsant [24,25], antimalarial [26], antitubercular [27,28] and kinase inhibitor [29,30]. It should be mentioned that some FDA-approved drugs involve a benzothiazole carrier scaffold ( Figure 1). They include quizartinib, a receptor tyrosine kinase inhibitor, flutemetamol, a diagnostic tool for Alzheimer disease, and riluzole, a drug for treatment of amyotrophic lateral sclerosis. On the other hand, chromone derivatives possess many different biological activities such as anticancer [31][32][33], antimicrobial [34], anti-inflammatory [35,36], antitubercular [37,38] and others activities [39][40][41], which have generated an interest in developing further this scaffold. The chromone scaffold is also present in some approved drugs such as flavoxate (Figure 2), a muscarinic antagonist and spasmolytic. This drug is used to treat urinary bladder spasms and is indicated for the symptomatic relief of conditions associated with lack of muscle control in the bladder, such as dysuria, urgency, and nocturia. On the other hand, chromone derivatives possess many different biological activities such as anticancer [31][32][33], antimicrobial [34], anti-inflammatory [35,36], antitubercular [37,38] and others activities [39][40][41], which have generated an interest in developing further this scaffold. The chromone scaffold is also present in some approved drugs such as flavoxate ( Figure 2), a muscarinic antagonist and spasmolytic. This drug is used to treat urinary bladder spasms and is indicated for the symptomatic relief of conditions associated with lack of muscle control in the bladder, such as dysuria, urgency, and nocturia. Taking into account these facts, we designed and synthesized new derivatives in corporating two pharmacophores, benzothiazole and chromone, in the frame of one molecule using the hybridization approach [42]. Hence, in the present study, the synthe sized hybrid molecules have been analyzed for their inhibitory potential against cance cell viability, especially on the colon cancer and cervical cancer cell lines. Further, to Taking into account these facts, we designed and synthesized new derivatives incorporating two pharmacophores, benzothiazole and chromone, in the frame of one molecule using the hybridization approach [42]. Hence, in the present study, the synthesized hybrid molecules have been analyzed for their inhibitory potential against cancer cell viability, especially on the colon cancer and cervical cancer cell lines. Further, to identify their potential in inhibiting the DDR signaling pathway, the compounds have also been subjected to assays that specifically inhibit ATR expression in these cancer cells.
The reaction of 2-unsubstituted chromones 2a or 2e [46] with hydroxylamine in pyridine afforded 2-aminochromones 5a or 5b as a result of subsequent ring-opening and ring-closure reactions. This unusual behavior of chromones 2a or 2e toward hydroxylamine can be explained by the strong electron-withdrawing effect of the aza-heterocyclic moiety in position 3 of the chromone ring. The bearing of ω-carboxyalkyl group 3-hetarylchromones 6a-6d and related 3-aryl-4-(2-benzothiazolyl)pyrazole 6e were synthesized as we have earlier reported [47] (Scheme 1).
Thus, in chromone Mannich bases 7 the singlet peak (except for compounds 7c, 7j-7l) of methylene group at 4.02-4.20 ppm was present as well as the secondary amine moiety. In the instance of piperidine and piperazine derivatives, in 1 H-NMR spectra signals of cyclic amine protons usually appear as wide non-resolved multiplets. In the case of chromones 7j-7l with 1,3,3-trimethyl-6-azabicyclo [3.2.1]octane moiety, we observed two doublets of diastereotopic 8-CH 2 group at 4.00-4.05 ppm and 4.28-4.32 ppm with heminal J 14.3-14.5 Hz. In 1 H-NMR peaks of 8-CH 2 group, we observed two multiplets at 3.90 and 4.33 ppm due to difficult conversion of the piperidine ring and/or the presence of stereocenter.

Cell Viability Studies-Initial Screening
In order to determine the effect of the compounds on cell viability, the studies were carried out using MTT assay in HCT116 and HeLa cell lines. Initial screening of the compounds was carried out at a final concentration of 10 µM. The cell viability was re-

Cell Viability Studies-Initial Screening
In order to determine the effect of the compounds on cell viability, the studies were carried out using MTT assay in HCT116 and HeLa cell lines. Initial screening of the compounds was carried out at a final concentration of 10 µM. The cell viability was reduced by ≤50% in the presence of three compounds, namely 2c, 7h and 7l, hence showing cell  Compounds 2c, 7h and 7l showed a significant reduction in cell viability for both the cervical cancer and colon cancer cell lines, suggesting that these compounds could be potent anticancer agents, especially for these two types of cancer. In order to identify the minimum concentration of these compounds required to reduce the cell viability of these cell lines by 50% or more, these compounds were further analyzed for their cytotoxicity effects using dose-response studies. The dose-response studies would be performed under similar conditions as those for the initial screening, for a concentration range of the potent compounds, so as to identify the half maximal inhibitory concentration (IC50) of each compound required to cause cell toxicity.

Cell Viability Studies-Dose Response
The dose-response studies were conducted using the MTT assay for 2c, 7h and 7l, for a dose range of 0-100 µM similar to the initial screening protocol. Further, the absorbance was measured at 570 nm using the PerkinElmer EnVision Multilabel Reader and the IC50 values calculated for all the three compounds (Table 1).  Compounds 2c, 7h and 7l showed a significant reduction in cell viability for both the cervical cancer and colon cancer cell lines, suggesting that these compounds could be potent anticancer agents, especially for these two types of cancer. In order to identify the minimum concentration of these compounds required to reduce the cell viability of these cell lines by 50% or more, these compounds were further analyzed for their cytotoxicity effects using dose-response studies. The dose-response studies would be performed under similar conditions as those for the initial screening, for a concentration range of the potent compounds, so as to identify the half maximal inhibitory concentration (IC 50 ) of each compound required to cause cell toxicity.

Cell Viability Studies-Dose Response
The dose-response studies were conducted using the MTT assay for 2c, 7h and 7l, for a dose range of 0-100 µM similar to the initial screening protocol. Further, the absorbance was measured at 570 nm using the PerkinElmer EnVision Multilabel Reader and the IC 50 values calculated for all the three compounds (Table 1).  Figure 5 represents the dose-response curves for the compounds 2c, 7h and 7l in HCT116 cells after 72 h incubation. Similarly, Figure 6 represents the dose-response curves for the compounds 2c, 7h and 7l in HCT116 cells after 72 h incubation.  Similarly, Figure 6 represents the dose-response curves for the compounds 2c, 7h and 7l in HCT116 cells after 72 h incubation.
Compounds 2c and 7h showed IC 50 values of 3.670 µM and 6.553 µM against HCT116 cell lines, while 7l showed an IC 50 value of 2.527 µM. However, all three compounds were equally potent against the HeLa cell line with IC 50 values of 2.642 µM, 3.995 µM and 2.659 µM, respectively. Further, to analyze the effect of these compounds specifically on the DDR signaling pathway, an essential pathway for cancer cell survival, the compounds were subjected to cell-based assays that would determine the inhibition of these pathways. Similarly, Figure 6 represents the dose-response curves for the compounds 2c, 7h and 7l in HCT116 cells after 72 h incubation.

Immunoblot Assay
The effect of the synthesized compounds to inhibit the cancer cell's DNA damage response (DDR) signalling pathway, a common pathway preferred by cancer cells to ensure genomic integrity, as well as cell viability [51], was analyzed by carrying out an immunoblot assay for one of the major kinases involved in the regulation of the pathway. Ataxia telangiectasia mutated and Rad-3 related (ATR) kinase, a member of the the PIKK (phosphatidylinositol-3-kinase-related kinases) family, are involved in the phosphorylation of Ser/Thr residues of its downstream substrate Chk1. The inhibition of Chk1 phosphorylation has been proved to be a clear indication of the inhibition of ATR signalling. Hence, the immunoblot assay helps in the determination of the inhibitory potential of the synthesized compounds against ATR kinase in the DDR pathway.
The analysis of inhibition of phosphorylation of Chk1 at Ser 317 in HeLa and HCT116 cells was carried out by immunoblotting using anti-pChk1 Ser 317 (rabbit) primary antibody and anti-rabbit IgG HRP-linked secondary antibody. Of the three compounds tested, compound 7h showed inhibition of ATR signaling which was observed by checking the status of ChK1 in HeLa cells (Figure 7). However, the results for HCT116 do not show significant inhibition of ATR signaling in the cases of compounds 7h and 2c, while 7l showed inhibition with respect to the control.
Further, to analyze the effect of the concentration of the compounds on the expression of Chk1 and pChk1 at Ser 317 in HeLa cells, immunoblotting assay using ChK1 mouse monoclonal antibody, and anti-pChk1 Ser 317 (rabbit) primary antibody were carried out. Anti-mouse IgG HRP-linked antibody and anti-rabbit IgG HRP-linked secondary antibody were used as secondary antibodies, respectively. The inhibition of Chk1 was observed at 2 µM and 5 µM concentrations of compound 7h. In the cases of compounds 2c and 7l, the ChK1 and pChK1 expression in the control lanes did not provide a clear insight into the inhibition of ATR signaling (Figure 8).
HCT116 cells was carried out by immunoblotting using anti-pChk1 Ser 317 (rabbit) primary antibody and anti-rabbit IgG HRP-linked secondary antibody. Of the three compounds tested, compound 7h showed inhibition of ATR signaling which was observed by checking the status of ChK1 in HeLa cells (Figure 7). However, the results for HCT116 do not show significant inhibition of ATR signaling in the cases of compounds 7h and 2c, while 7l showed inhibition with respect to the control.

Molecular Docking
The use of molecular docking as a successful computational tool in drug disco to model and visualize the interactions between small molecules and their specif gets, has been prevalent since the 1980s. The conformations and binding intera corresponding to the behavior of the small molecules at the binding site of the drug have also been an essential factor in identifying the mechanism of enzyme inhib which further enhance the drug development processes [52].
Molecular docking resulted in an in-depth analysis of the major binding intera between the compounds with the ATR kinase domain. Compound 2c showed the GScore and DockScore of −5.5 kcal/mol, followed by a GScore and DockScore o kcal/mol and −5.1 kcal/mol for Torin2. Compound 7l showed the GScore and Dock of −5.2 kcal/mol and −4.8 kcal/mol. This was followed by compound 7h with a G and DockScore of −4.3 kcal/mol and −3.4 kcal/mol ( Table 2).

Molecular Docking
The use of molecular docking as a successful computational tool in drug discovery, to model and visualize the interactions between small molecules and their specific targets, has been prevalent since the 1980s. The conformations and binding interactions corresponding to the behavior of the small molecules at the binding site of the drug target have also been an essential factor in identifying the mechanism of enzyme inhibition, which further enhance the drug development processes [52].
Molecular docking resulted in an in-depth analysis of the major binding interactions between the compounds with the ATR kinase domain. Compound 2c showed the same GScore and DockScore of −5.5 kcal/mol, followed by a GScore and DockScore of −5.2 kcal/mol and −5.1 kcal/mol for Torin2. Compound 7l showed the GScore and DockScore of −5.2 kcal/mol and −4.8 kcal/mol. This was followed by compound 7h with a GScore and DockScore of −4.3 kcal/mol and −3.4 kcal/mol (Table 2). Two-dimensional ligand interaction diagrams show the major binding interactions between the compounds and the binding site of the ATR kinase domain. Torin2, the standard ligand had hydrogen bonding and π-π stacking interactions as the major interactions at the binding site. Compound 2c had the highest docking score of −5.5 kcal/mol, wherein hydrogen bonding, π-π stacking and π-cation interactions played the major role. Compound 7h had similar interactions in comparison to Torin2, with a lowest docking score of −3.4 kcal/mol. However, unlike all the ligands in the molecular docking study, compound 7l had the highest number of diverse interactions at the binding site, including hydrogen bonding, π-π stacking, π-cation interactions and salt bridge formation. The ligand interaction diagrams for the binding interactions of compounds 2c, 7h and 7l along with Torin2 are shown in Figure 9. The three-dimensional view of the ligand conformations in the binding site is crucial to visualize the interactions determined through ligand interaction diagrams. The binding poses of the compounds, in comparison to the standard ligand helps in deciphering the nature of binding of these compounds at the binding site. The 3D diagrams indicating the binding poses of the compounds 2c (pink), 7h (yellow), 7l (orange) and Torin2 (green) are depicted in Figure 10. The three-dimensional view of the ligand conformations in the binding site is crucial to visualize the interactions determined through ligand interaction diagrams. The binding poses of the compounds, in comparison to the standard ligand helps in deciphering the nature of binding of these compounds at the binding site. The 3D diagrams indicating the binding poses of the compounds 2c (pink), 7h (yellow), 7l (orange) and Torin2 (green) are depicted in Figure 10.

General Procedure for the Synthesis of 2-Ethoxycarbonyl Chromones 4a-4c
To a solution of the corresponding starting compounds 1a,b,d (5 mmol) in dry pyridine (10 mL) ethyl oxalyl chloride (15 mmol) was added dropwise under cooling. The reaction mixture was stirred for 24-48 h at room temperature and poured into 200 mL of 1N HCl. The formed precipitate was filtered off, dried, and re-crystallized from EtOH to furnish the desired products 4a-4c. A mixture of chromone 2a,e (1 mmol) and hydroxylamine hydrochloride (2 mmol) in pyridine (5 mL) was refluxed for 6-8 h. The reaction mixture was cooled, and the formed precipitate was filtered off, dried, and re-crystallized from EtOH to furnish the desired products 5a,b.

Cell Viability Studies
A set of 25 compounds was tested for cell viability studies using MTT assay, in HCT116 and HeLa cell lines. All experiments were carried out in triplicate as per previously reported methods [53,54]. For the viability studies, HCT116 and HeLa cells were seeded at the count of 7 × 10 3 cells per well in a transparent 96-well plates, and incubated for 24 h at 37 • C, in a humidified incubator with 5% CO 2 . Following this, the cells were treated with the compounds at a final concentration of 10 µM and incubated further for 72 h at 37 • C. After incubation, MTT [3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl tetrazolium bromide] was added at a final concentration of 0.5 mg/mL. The cells were further incubated for 4 h at 37 • C in an incubator. The formazan crystals formed were dissolved using 50 µL of DMSO. Thereafter, the absorbance was measured at 570 nm using the PerkinElmer EnVision Multilabel Reader (Perkin Elmer, Inc., Waltham, MA, USA). The cells treated with DMSO were used as a control for all the experiments. Both the cell lines showed viability ≤ 50% for three compounds in the initial screening, namely 2c, 7h and 7l. Hence, these three compounds were selected from the initial screening and were sensitized against HCT116 and HeLa, across a dose range of 0-100 µM by performing another cell viability assay. Finally, the results obtained were plotted using Graphpad Prism 8.0.2 to obtain the respective IC 50 values.

Immunoblot Assay
The immunoblot assay was carried out as per previous reports [2]. HCT116 and HeLa cells were seeded at the count of 0.5 × 10 5 cells per ml in a 6-well plate and incubated overnight at 37 • C, in a humidified CO 2 incubator. For this assay, the cells were treated with the hit compounds with their concentration range approximately equal to their IC 50 values (Table 3), and incubated for 1 h at 37 • C. After the incubation, the cells were given UV radiation energy via UVP cross linker at 50 mJ/cm 2 and further incubated for 1 h at 37 • C. The media was stored before UV radiation and added back after the treatment. The cells were then trypsinized, washed with ice cold 1X PBS, and lysed with RIPA buffer. The cell lysates were isolated by centrifugation at 4 • C, 13,500 rpm for 10 min. The protein concentration was normalized by Bradford assay. The samples were loaded on SDS-PAGE gel and subjected to immunoblotting. β-actin was taken as a loading control in the experiment. Further, to identify the pattern of inhibition of Chk1 phosphorylation, the immunoblot assay was repeated for a range of concentrations of the compounds 2c, 7h, and 7l in HeLa cell line against Chk1 and pChk1 antibodies. The experiments were conducted in a similar way for a concentration range of 2 µM, 5 µM and 10 µM. β-actin was taken as a loading control in the experiment.

Molecular Docking
In the present study, the compounds 2c, 7h and 7l were subjected to molecular docking studies at the Torin2 binding site of homology modelled ATR kinase domain [55], using the Glide module (XP) of Maestro 12.7.156 version of Schrödinger software [56]. The homology modelled ATR kinase domain was further prepared for molecular docking, using the Protein Preparation Wizard by including Epik state penalties (pH 7.0 ± 2.0), and by performing the minimization using 0.3 Å RMSD and OPLS4 force field [57][58][59]. Further, the compounds 2c, 7h and 7l were included in the project using the 2D sketcher option in the software, following which the 3D structures were incorporated in the workspace. The standard molecule for the study was selected to be Torin2, the standard inhibitor in the modelled structure, which is a well-known mTOR and ATR/ATM inhibitor known for its potency against the p-Chk1 Ser 317 and p70 S6K Thr 389 substrates in the DDR pathway through cell studies. The ligands were then prepared using the LigPrep module [57].
The ligand preparation was followed by receptor grid generation, wherein the grid was generated using the Glide module, by selecting the Torin2 ligand from the minimized protein structure. Ligand docking for the standard molecule and the synthesized compounds was conducted in the Glide XP (Extra Precision) module [56].
The docking results were analyzed in the XP Visualizer tool, wherein the binding energies were analyzed using the docking score instead of GlideScore, since the Epik state penalties were included during the protein and ligand preparations. The best pose of each of the compounds 2c, 7h and 7l with higher negative value of DockScore were considered for the study.

Conclusions
A series of benzothiazole and chromone derivatives were synthesized and evaluated for their anticancer activity as inhibitors of ATR kinase. Cell viability of a set of 25 compounds was performed using MTT assay in HCT116 and HeLa cell lines, involving 72 h incubation of the compounds at a final concentration of 10 µM. Cells incubated with compounds 2c, 7h and 7l were found to show a viability ≤50, and were taken forward for dose-response studies. The three compounds showed IC 50 values in micromolar range. Among all the compounds tested, 7l had the best IC 50 values in both the cell lines. Compounds 2c and 7l were found to be equally cytotoxic towards both the cell lines, namely, HCT116 and Hela, while compound 7h showed better cytotoxicity towards HeLa cell line.
To further identify the inhibitory potential of the synthesized compounds in the DNA damage response (DDR) signalling pathway, an immunoblot assay was carried out for these three compounds in order to analyze the inhibition of phosphorylation of Chk1 at Ser 317 in HeLa and HCT116 cells. Compound 7h showed inhibition of pChk1 at Ser 317 in HeLa cells at a concentration of 3.995 µM. Further analysis for Chk1 and pChk1 expression was analyzed in Hela cells by treatment against all the three compounds at a range of concentrations such as 2, 5 and 10 µM, wherein compound 7h showed Chk1 inhibition at 2 and 5 µM, while pChk1 expression was observed for compound 7l at a concentration of 5 µM. The binding interactions of the compounds with the ATR kinase domain were studied through molecular docking, wherein compound 2c, 7h and 7l showed binding interactions similar and/or lesser than the standard ligand Torin2. Thus, these compounds can serve as starting point for further modifications in order to improve the activity.